Liposome containing hydrophobic iodine compound

ABSTRACT

A liposome containing a hydrophobic iodine compound such as a 1,3,5-triiodobenzene derivative having at least one substituent containing 18 or more carbon atoms as a membrane component, and an X-ray contrast medium containing the liposome for use in radiography of a vascular disease and the like.

TECHNICAL FIELD

The present invention relates to a liposome. More specifically, theinvention relates to a liposome that can be utilized for a method forselectively accumulating a hydrophobic iodine compound in a pathologicallesion and imaging said lesion in contrast to non-pathological site.

BACKGROUND ART

In the modern society, especially in the societies of advancedcountries, opportunities of ingesting high calorie and high fat diet areincreasing. For this reason, mortalities due to ischemic diseasesresulting from arteriosclerosis (heart diseases such as myocardialinfarction and angina pectoris, cerebrovascular diseases such ascerebral infarction and cerebral hemorrhage) have been increasing.Therefore, it has been desired to diagnose such conditions at an earlystage to employ an appropriate treatment. However, no satisfactorymethod is available for diagnosing progress of arteriosclerosis at anearly stage before the onsets of the aforementioned diseases.

Methods for diagnosing arteriosclerosis are basically classified intonon-invasive methods and invasive methods in which a catheter or thelike is inserted into an artery. Among them, typical non-invasivemethods include X-ray angiography and ultrasonography. However, by thesemethods, it is almost impossible to detect arteriosclerosis at an earlystage, especially constriction of coronary artery, which causesmyocardial infarction or angina pectoris, at an early stage before theonset of these diseases.

CT, MRI and the like may sometimes be used as another class ofnon-invasive methods. However, these methods have been mainly developedfor detection of tumors, and accordingly, they have a problem of a lowresolution of arteriosclerotic lesions. In addition, the methods requireexpensive and large-scale apparatuses, which limits employable hospitalsand general applicability. Further, methods utilizing radioisotopes havealso been investigated. However, these methods still remain at anexperimental level.

As the invasive methods, intravascular echo, vascular endoscope and thelike have been used. It is recognized that an arteriosclerotic lesionwith a thickness as thin as 0.1 mm can be measured by these methods.However, for employment of these methods, it is necessary to arteriallyinsert an ultrasonic oscillator or an endoscope attached to an end of acatheter, which may result in serious physical stress and heaviness aswell as a risk of a patient. Therefore, although these methods have beenused therapeutically for patients after the attack of myocardialinfarction and the like or as secondary prophylaxis, they cannot be usedfor a diagnostic purpose to know as to presence or absence or a degreeof progress of arteriosclerosis in a patient before onset.

Among the aforementioned methods, a method most widely used foridentification of a lesion of arterial vasoconstriction is X-rayangiography. This method comprises the step of administration of awater-soluble iodine contrast medium to visualize vascular flows, anddetecting a lesion at which the flows are obstructed. However, thesemethods can only detect a lesion where constriction progresses 50% ormore and fail to detect a lesion before the onset of attack of anischemic disease.

Separately from the above methods, attempts have also been reported inwhich a hydrophobic iodine contrast medium or a hydrophilic contrastmedium is formulated for selective accumulation in a target lesion(International Patent Publications WO95/19186, WO95/21631, WO89/00812,British Patent No. 867650, WO96/00089, WO94/19025, WO96/40615,WO95/2295, WO98/41239, WO98/23297, WO99/02193, WO97/06132, U.S. Pat.Nos. 4,192,859, 4,567,034, 4,925,649, Pharm. Res., 16 (3), 420 (1999),J. Pharm. Sci., 72 (8), 898 (1983), Invest. Radiol., 18 (3), 275 (1983).For example, Pharm. Res., 16 (3), 420 (1999) discloses that, byinjection of an oil-particle dispersion of cholesteryl iopanoate as ahydrophobic compound, the iodine compound accumulates inarteriosclerotic lesions of experimental animals.

Further, J. Pharm. Sci. 72 (8), 898 (1983) discloses examples of X-rayhepatography and splenography by injection of an oil-particle dispersionof cholesteryl iopanoate. U.S. Pat. No. 4,567,034 describes a method ofselective hepatography or splenography utilizing liposomes encapsulatingan ester of diatrizoic acid. International Patent PublicationsWO96/28414 and WO96/00089 disclose contrast media for imaging vascularpools or lymphatic systems. However, the methods using theseformulations are not satisfactory in efficiency and selectivity for apurpose of selective contrast of vascular diseases, and no examplethereof is reported in which vascular diseases are imaged by utilizingX-ray irradiation.

Mechanisms of onset of arterial diseases have recently beenprogressively elucidated at levels of genes, proteins and cells (J.Biol. Chem., 1996, 271 (44) 27346–52; Nature, 386 (6662) 292–6). As forarteriosclerosis, it has been elucidated that plural kinds of cells formlesions while they mutually control their proliferation (Arterioscler.Throm. Vasc. Biol., 1999 (3) 461–71; Lab Invest 1998, 78 (4) 423–34).However, no example has been demonstrated which reproduces the state ofa lesion, in which plural kinds of cells are involved as mentionedabove, in a cell culture vessel, and accordingly, evaluation of drugsfor arteriosclerosis or restenosis have so far been conducted mainly byusing model animals.

However, such methods using animals are time-consuming and require ahigh cost, and their use is also required to be minimum from a viewpointof prevention of cruelty to animals. Therefore, an in vitro evaluationmethod of reproducing a state of arteriosclerotic lesion has beendesired for screening of a large number of compounds for a short periodof time.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide means for selectivelyaccumulating an iodine compound in a lesion of a vascular disease causedby abnormal proliferation of vascular smooth muscle cells such asarteriosclerosis and restenosis after PTCA. Another object of thepresent invention is to provide a means for imaging of a biologicalenvironment of a vascular disease or the like by X-ray radiography usingthe aforementioned means. The inventors of the present inventionconducted various studies to achieve the foregoing objects, and as aresult, they found that liposomes containing a hydrophobic iodinecompound as one of membrane components accumulated in vascular smoothmuscle cells and foam macrophages, which are main components ofarteriosclerotic lesion.

A further object of the present invention is to provide a cell culturesystem reproducing a state of lesion of arteriosclerosis, restenosis orthe like and a method for producing such a cell culture system. Anotherobject of the present invention is to provide a method for evaluating adrug for arterial diseases by using the aforementioned means. Theinventors of the present invention conducted various studies to achievethe foregoing objects, and as a result, they found that a cell culturesystem reproducing a state of lesion of arteriosclerosis, restenosis orthe like was successfully provided by simultaneously culturing in asingle cell culture vessel two or more kinds of cell species that form alesion of a mammalian disease. The present invention was achieved on thebasis of these findings.

The present invention thus provides a liposome containing a hydrophobiciodine compound as a membrane component. As preferred embodiments of thepresent invention, provided are the aforementioned liposome, wherein thehydrophobic iodine compound is a 1,3,5-triiodobenzene derivative havingat least one substituent containing 18 or more carbon atoms; theaforementioned liposome, which contains a lipid selected from the groupconsisting of phosphatidylcholines and phosphatidylserines as a membranecomponent; the aforementioned liposome, which contains a phosphoric aciddialkyl ester which is a diester of an alkyl containing 6 or more carbonatoms as a membrane component; and the aforementioned liposome, whereinthe substituent containing 18 or more carbon atoms is a residue of acholesterol derivative.

From another aspect, the present invention provides an X-ray contrastmedium, which comprises the aforementioned liposome. As preferredembodiments of the invention, provided are the aforementioned X-raycontrast medium, which is used for radiography of a vascular disease,preferably radiography of vascular smooth muscle cells which areabnormally proliferated under an influence of foam macrophages, forexample, for radiography of an arteriosclerotic lesion or restenosisafter PTCA. The present invention also provides a method for contrastinga vascular disease, which comprises a step of X-ray radiography usingthe aforementioned liposome, and use of the aforementioned liposome forthe manufacture of the aforementioned X-ray contrast medium.

From a further aspect, the present invention provides a method forpreparing a cell culture system, which comprises a step ofsimultaneously culturing, in a single cell culture vessel, two or morekinds of cell species which are involved in formation of a lesion of amammalian disease. As preferred embodiments of the invention, providedare the aforementioned method, wherein the cell species comprise primaryculture cells isolated from a living body or established subculturecells; the aforementioned method, wherein the cell species comprisemammalian primary culture cells; and the aforementioned method, whereinthe mammalian disease is a human disease.

As more preferred embodiments of the above invention, provided are theaforementioned method, wherein at least one of the cell species consistsof cells involved in formation of arteriosclerotic lesion; theaforementioned method, wherein the cell species are selected from thegroup consisting of macrophages, vascular smooth muscle cells, andvascular endothelial cells; the aforementioned method, which comprises astep of culturing two kinds of cell species, in a single culture vessel,under being partitioned off by means of a cell filter; and theaforementioned method, wherein the two kinds of cell species consist ofmacrophages and vascular smooth muscle cells. As a particularlypreferred embodiment of the present invention, provided is a method forproducing a cell culture system that reproduces a state of anarteriosclerotic lesion, which comprises simultaneously culturing in asingle culture vessel two kinds of mammalian primary culture cellsinvolved in formation of arteriosclerotic lesion under being partitionedoff by mean of a cell filter.

The present invention further provides a cell culture system, which isobtainable by the aforementioned method for producing a cell culturesystem. The present invention also provides a method for screening adrug by using a cell culture system that is obtainable by theaforementioned method for producing a cell culture system. As preferredembodiments, the present invention provides a method for determiningeffectiveness of a drug on a vascular disease, which comprisesdetermining action of the drug on vascular smooth muscle cellsproliferated by culturing foam macrophages and the vascular smoothmuscle cells under being partitioned off by means of a cell filter; anda method for evaluating permeation ability of a drug into a lesion ofvascular disease, which comprises measuring action of the drug onvascular smooth muscle cells proliferated by culturing foam macrophagesand the vascular smooth muscle cells under being partitioned off bymeans of a cell filter.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows results of induction of mouse vascular smooth muscle cellproliferation in the presence of foam mouse macrophages.

FIG. 2 shows a proliferation curve of mouse vascular smooth muscle cellsobtained without addition of foam mouse macrophages.

FIG. 3 shows results of induction of rat vascular smooth muscle cellproliferation in the presence of foam rat macrophages.

FIG. 4 shows results of induction of rabbit vascular smooth muscle cellproliferation in the presence of foam rabbit macrophages.

FIG. 5 shows uptake of liposomes by mouse vascular smooth muscle cells.In the figure, (1) shows results obtained by adding a liposomepreparation after 3 days to the culture system of FIG. 2 not added withfoam macrophages, and then continuing the culture for 1 day, (2) showsresults obtained by adding a liposome preparation after 5 days to theculture system of FIG. 1 which was added with foam macrophages, and thencontinuing culture for 1 day, and (3) shows results obtained by adding aliposome preparation after 7 days to the culture system of FIG. 1 whichwas added with foam macrophages, and then continuing culture for 1 day.

FIG. 6 shows the results obtained by using an oil-particle dispersioninstead of the liposomes of the present invention.

FIG. 7 shows uptake of liposomes by rat vascular smooth muscle cellscultured under the culture conditions mentioned in FIG. 3. In thefigure, (1), (2) and (3) have the same meanings as those mentioned inFIG. 5.

FIG. 8 shows uptake of liposomes by rabbit vascular smooth muscle cellscultured under the culture conditions mentioned in FIG. 4. In thefigure, (1), (2) and (3) have the same meanings as those mentioned inFIG. 5.

FIG. 9 depicts photographs showing the results of X-ray radiography ofarteriosclerotic lesions by using the liposomes of the presentinvention. In the figure, “before administration” indicates the resultobtained before administration of liposomes, and “after administration”indicates the result obtained immediately after administration ofliposomes.

FIG. 10 depicts photographs showing the results of X-ray radiography ofarteriosclerotic lesions using the liposomes of the present invention.In the figure, “15 minutes after administration” indicates the resultobtained 15 minutes after administration of liposomes, and “30 minutesafter administration” indicates the result obtained 30 minutes afteradministration of liposomes.

BEST MODE FOR CARRYING OUT THE INVENTION

A type of the hydrophobic iodine compound is not particularly limited.For example, an iodobenzene derivative is preferred. More preferred is a1,3,5-triiodobenzene derivative having at least one substituent having18 or more carbon atoms. The substituent having 18 or more carbon atomsis preferably a hydrophobic group for stable localization of the1,3,5-triiodobenzene residue, as being an iodine contrasting moiety, ina bilayer of the liposome. For example, preferred is a substituenthaving 20 or more carbon atoms in which the total number of oxygen atomand nitrogen atom is 10 or less. The hydrophobic substituent morepreferably has a structure similar to that of lipid componentsconstituting biological membranes. Preferred examples of a hydrophobiciodine compound that satisfies such conditions include, for example,1,3,5-triiodobenzene derivatives having a cholesterol derivative residueas a substituent disclosed in J. Med. Chem., 25 (12), 1500 (1982);Steroids, 49 (6), 531 (1987); Pharm. Res., 6 (12), 1011 (1989);International Patent Publications WO95/19186, WO96/28414 and the like.

As the cholesterol derivatives, those described in the aforementionedpublications are preferred, and cholesterol is particularly preferred.Preferred are compounds in which cholesterol is bound to a hydrophobiciodine compound, such as 1,3,5-triiodobenzene, via the 3-hydroxyl group.For binding of the hydroxyl group of cholesterol and the hydrophobiciodine compound such as 1,3,5-triiodobenzene, for example, means such asan ester bond, ether bond, urethane bond, and carbonic acid ester bondcan be used. An ester bond is preferred. Cholesterol and the hydrophobiciodine compound such as 1,3,5-triiodobenzene may be directly bound viaany of the aforementioned bonds, or bound via an appropriate bridginggroup. Examples of suitable bridging group include a linear or branchedalkylene group having 5 or less carbon atoms.

The hydrophobic iodine compound, preferably 1,3,5-triiodobenzenecompound, may have one or more substituents other than theaforementioned substituent having 18 or more carbon atoms. The type,substituting position and numbers of the substituents are notparticularly limited. For example, it is preferred that a substituted orunsubstituted amino group, a substituted or unsubstituted acylaminogroup, a hydroxyl group, a carboxyl group or the like substitutes on thebenzene ring of the hydrophobic iodine compound. Preferred substituentsare a substituted or unsubstituted amino group and a substituted orunsubstituted acylamino group. Examples of the amino group having asubstituent include a monoalkylamino group, a dialkylamino group and thelike, and examples of the acylamino group having a substituent includetrifluoroacetylamino group, p-chlorobenzoylamino group and the like.

Preferred examples of the hydrophobic iodine compound are listed below.However, the liposomes of the present invention are not limited to thosecontaining these compounds.

The hydrophobic iodine compound is contained as a component of amembrane of the liposome, and a content of the compound in the liposomeis about from 10 to 90 mass %, preferably from 10 to 80 mass %, morepreferably from 20 to 80 mass %, based on the total mass of the membranecomponents of the liposome. One kind of the hydrophobic iodine compoundmay be used as a membrane component, or two or more kinds of thehydrophobic iodine compounds may be used in combination.

As other membrane components constituting the liposome, any of lipidcompounds ordinarily used for preparation of liposomes can be used. Forexample, such compounds are described in Biochim. Biophys. Acta, 150(4), 44 (1982); Adv. in Lipid. Res., 16 (1) 1 (1978); “RESEARCH INLIPOSOMES”, P. Machy, L. Leserman, John Libbey EUROTEXT Co.; “Liposome”(Ed., Nojima, Sunamoto and Inoue, Nankodo) and the like. As the lipidcompounds, phospholipids are preferred, and phosphatidylcholines (PC)are particularly preferred. Preferred examples of phosphatidylcholinesinclude, but not limited thereto, egg PC, dimyristoyl-PC (DMPC),dipalmitoyl-PC (DPPC), distearoyl-PC (DSPC), dioleyl-PC (DOPC) and thelike.

According to a preferred embodiment of the present invention, aphosphatidylcholines and a phosphatidylserine (PS) can be used incombination. Examples of the phosphatidylserines include those havinglipid moieties similar to those of the phospholipids mentioned aspreferred examples of the phosphatidylcholines. When aphosphatidylcholine and a phosphatidylserine are used in combination,molar ratio of PC and PS (PC:PS) used is preferably in the range of90:10 to 10:90, further preferably 30:70 to 70:30.

Another preferred embodiment of the liposome of the present inventionincludes the liposome containing a phosphatidylcholine and aphosphatidylserine and further containing a phosphoric acid dialkylester as membrane components. The two alkyl groups constituting thedialkyl ester of phosphoric acid are preferably the same groups. Eachgroup may contain 6 or more carbon atoms, preferably 10 or more carbonatoms, more preferably 12 or more carbon atoms. Preferred examples ofthe phosphoric acid dialkyl ester include, but not limited thereto,dilauryl phosphate, dimyristyl phosphate, dicetyl phosphate and thelike. In this embodiment, preferred amount of the phosphoric aciddialkyl ester is from 1 to 50 mass %, preferably from 1 to 30 mass %,further preferably from 1 to 20 mass %, based on the total mass ofphosphatidylcholine and phosphatidylserine.

In the liposome containing a phosphatidylcholine, a phosphatidylserine,a phosphoric acid dialkyl ester and a hydrophobic iodine compound asmembrane components, preferred weight ratio of PC, PS, phosphoric aciddialkyl ester and hydrophobic iodine compound may be chosen from 5 to 40mass %: from 5 to 40 mass %: from 1 to 10 mass %: from 15 to 80 mass %.

The components of the liposome of the present invention are not limitedto the aforementioned four kinds of compounds, and other components maybe added. Examples of such components include cholesterol, cholesterolesters, sphingomyelin, monosial ganglioside GM1 derivatives described inFEBS Lett., 223, 42 (1987); Proc. Natl. Acad. Sci., USA, 85, 6949 (1988)etc., glucuronic acid derivatives described in Chem. Lett., 2145 (1989);Biochim. Biophys. Acta, 1148, 77 (1992) etc., polyethylene glycolderivatives described in Biochim. Biophys. Acta, 1029, 91 (1990); FEBSLett., 268, 235 (1990) and the like. However, the components are notlimited to these examples.

The liposome of the present invention can be prepared by any methodsknown in the field of the art. Examples of the preparation method aredescribed in the references as general review of liposomes, which arementioned above, as well as in Ann. Rev. Biophys. Bioeng., 9, 467(1980), “Liopsomes” (Ed. by M. J. Ostro, MARCELL DEKKER, INC.) and thelike. Specific examples include, but not limited thereto, theultrasonication method, ethanol injection method, French press method,ether injection method, cholic acid method, calcium fusion method,freeze and thawing method, reverse phase evaporation method and thelike. Size of the liposome of the present invention may be any of thoseobtainable by the aforementioned methods. Generally, a size in averagemay be 400 nm or less, preferably 200 nm or less. Structure of theliposome is not particularly limited, and may be unilamellar ormultilamellar structure. It is also possible to formulate one or morekinds of appropriate drugs or other contrast media in the liposome.

When the liposomes of the present invention are used as a contrastmedium, they can be preferably administered parenterally, morepreferably administered intravenously. For example, preparations in theform of an injection or a drip infusion can be provided as powderycompositions in a lyophilized form, and they can be used by beingdissolved or resuspended just before use in water or an appropriatesolvent (e.g., physiological saline, glucose infusion, bufferingsolution and the like). When the liposomes of the present invention areused as a contrast medium, the dose can be suitably determined so thatan iodine content in the liposomes becomes similar to that of aconventional iodine-containing contrast medium.

Although it is not intended to be bound by any specific theory, it isknown that, in vascular diseases such as arteriosclerosis or restenosisafter PTCA, vascular smooth muscle cells constituting tunica media ofblood vessel abnormally proliferate and migrate into endosporium at thesame time to narrow blood flow passages. Although triggers that initiatethe abnormal proliferation of normal vascular smooth muscle cells havenot yet been clearly elucidated, it is known that migration ofmacrophages into endosporium and foaming are important factors. It isreported that vascular smooth muscle cells then cause phenotypeconversion (from constricted to composite type).

When the liposomes of the present invention are used, the hydrophobiciodine compound can be selectively taken up into the vascular smoothmuscle cells abnormally proliferated under influences of foammacrophages. As a result, radiography becomes possible with highcontrast between a lesion and a non-pathological site. Therefore, thecontrast medium of the present invention can be suitably usedparticularly for X-ray radiography of vascular diseases. For example,radiography of arteriosclerotic lesion or restenosis after PTCA can beperformed. Method for imaging is not particularly limited. Imaging canbe performed by, for example, a method utilizing X-ray irradiation,radionuclide imaging utilizing an radioactive iodine-labeled compoundand the like. However, the method is not limited to these.

The method for producing a cell culture system provided by the presentinvention is characterized to comprise a step of culturing, in a singlecell culture vessel, two or more kinds of cell species which areinvolved in formation of lesion of a mammalian disease. As the cellspecies, primary culture cells and established subculture cells arepreferred, and mammalian primary culture cells are particularlypreferred. Preferred mammals include, but not limited thereto, human,dog, cat, pig, miniature pig, rabbit, hamster, rat, mouse and the like.Type of the culture vessel is not particularly limited, and for example,culture flask, culture test tube, dish, microplate and the like can besuitably used.

The two or more kinds of different cell species cultured in a singleculture vessel may be derived from homologous animals or heterogenousanimals. They may preferably be derived from homologous animals. In thespecification, the term “involved in formation of a lesion” should beconstrued in its broadest sense, including a condition where pluralkinds of cells form a lesion under mutual control of proliferation andthe like, and the term should not be construed in any limitative sense.As the different cell species, it is preferred to chose cell speciesdifferent from cytobiological y or cytotaxonomical viewpoint. Morepreferably, two kinds of cell species may be chosen that coexist in acertain lesion and form the lesion under mutual control ofproliferation. For example, macrophages and vascular smooth musclecells, which coexist in an arteriosclerotic lesion, are preferablychosen as the two kinds of cell species.

In a preferred embodiment of the method for preparing a cell culturesystem of the present invention, the two kinds of cell species can becultured in a single well under being partitioned off by means of a cellfilter. According to another preferred embodiment of the presentinvention, the two kinds of cell species used are preferably cells thatform an arteriosclerotic legion. Preferred cell species includemacrophages, vascular smooth muscle cells, vascular endothelium cells, Tcells, mast cells and the like. Among them, preferred cell speciesinclude macrophages, vascular smooth muscle cells and vascularendothelium cells, and particularly preferred cell species include acombination of macrophages and vascular smooth muscle cells. In thecombination, the macrophages are preferably made into foam cellsbeforehand.

According to the present invention, a method is provided for evaluationof permeation ability of a drug into a lesion of a vascular disease,which comprises a step of measuring action of the drug on vascularsmooth muscle cells which are proliferated by culturing foam macrophagesand the vascular smooth muscle cells under being partitioned off bymeans of a cell filter. The term “action of drug” used in thespecification should be construed in its broadest sense includingtherapeutic effect, diagnostic effect and the like. Types of the cellfilter are not particularly limited so long as they have such a poresize that does not allow foam macrophages and vascular smooth musclecells to pass through pores. For example, as a pore size that does notallow the cells to pass through pores, a filter having a pore size of0.4 μm or less can be used. Depending on types of foam macrophages andcells to be used, a cell filter having an appropriate pore size can beeasily chosen.

Although it is not intended to be bound by any specific theory, whenfoam macrophages and vascular smooth muscle cells are cultured underbeing partitioned off by means of a cell filter, a proliferationactivating substance derived from the foam macrophages acts on thevascular smooth muscle cells to induce their proliferation, and thusabnormal proliferation of vascular smooth muscle cells in a vasculardisease such as arteriosclerosis and restenosis after PTCA can bereproduced in vitro. Investigation of uptake of drugs into theproliferated vascular smooth muscle cells enables screening of a drughaving a high effectiveness on vascular diseases such asarteriosclerosis and restenosis after PTCA.

EXAMPLES

The present invention will be explained more specifically with referenceto the examples. However, the scope of the present invention is notlimited to the following examples.

Example 1 Preparation of Culture System of Vascular Smooth Muscle Cellsof which Proliferation is Activated by Foam Macrophages (1)

Vascular smooth muscle cells were isolated from mouse aorta endothelium(“Tissue Culture Method”, 10th Edition, ed. by the Japanese TissueCulture Association, published by Kodansha, 1998). The isolated vascularsmooth muscle cells were suspended in 10% FBS Eagle's MEM (GIBCO, No.11095-080) and inoculated in wells of a 12-well microplate (FALCON, No.3503). The number of the cells in each well was adjusted to 10,000cells. The cells were cultured for 3 days under conditions of 37° C. and5% CO₂.

Then, foam mouse peritoneal macrophages were prepared according to themethod described in Biochimica Biophysica Acta, 1213, 127 (1994).200,000 cells of the foam macrophages were separated and inoculated onan insert cell (FALCON, No. 3180) placed on each well of the microplatewhere the vascular smooth muscle cells were cultured on the bottomsurface. The cells were cultured for 5 days under conditions of 37° C.and 5% CO₂. The cell numbers of the vascular smooth muscle cells in theabove experiment are shown in FIG. 1. Although the vascular smoothmuscle cells gently proliferated at an early stage after the start ofthe culture, they actively proliferated after the addition of the foammacrophages after 3 days and a subsequent induction period of about 1day. A proliferation curve of vascular smooth muscle cells not addedwith the macrophages is shown in FIG. 2. Comparison of the results shownin FIGS. 1 and 2 clearly indicates activating effect of the foammacrophages on the proliferation.

Example 2 Verification of Expression of Scavenger Receptors on VascularSmooth Muscle Cells

It is known that vascular smooth muscle cells in an arterioscleroticlesion express scavenger receptors on their surfaces to take up oxidizedLDL (Biochem. Phamacol., 15:57 (4), 383–6 (1999); Exp. Mol. Pathol., 64(3), 127–45, 1997). The vascular smooth muscle cells of the culturesystem of FIG. 1 were immunostained by using mouse scavenger receptorantibodies. As a result, although the expression was not observed on thevascular smooth muscle cells on the 3rd day from the inoculation, clearstaining was observed on the 6th day from the inoculation. When the foammacrophages on the cell filter were also similarly immunostained, clearstaining was also observed.

Example 3 Uptake of Oxidized LDL by Vascular Smooth Muscle Cells

In the culture system of FIG. 1, ¹²⁵I-labeled oxidized LDL was added tothe medium for the vascular smooth muscle cells on the 3rd day and 6thday from the inoculation. ¹²⁵I taken up into the cells was counted 24hours after each addition. The results are shown in Table 1. Cleardifference in uptake amount was observed between the results on the 3rdday and 6th day. The above results indicate that the vascular smoothmuscle cells cultured in the cell culture system of the presentinvention had properties similar to those of smooth muscle cells in alesion of arteriosclerosis, restenosis or the like.

TABLE 1 Day Uptake of ¹²⁵I-oxLDL 3rd Day from inoculation 0.52 ± 0.116th Day from inoculation 2.2 ± 0.4 ×10,000 cpm

Example 4 Preparation of Culture System of Vascular Smooth Muscle Cellsof which Proliferation is Activated by Foam Macrophages (2)

Culture systems comprising rat and rabbit macrophages and vascularsmooth muscle cells were prepared in the same manner as in Example 1.The cell numbers of vascular smooth muscle cells are shown in FIGS. 3and 4. In each of the experiments, a result similar to that of FIG. 1for mouse was obtained.

The vascular smooth muscle cells cultured in the culture system in FIG.1 had properties of healthy blood vessel until the 3rd day from theinoculation and properties of smooth muscle cells in an arterioscleroticlesion on and after the 7th day from the inoculation. Accordingly, ascreening of a drug selective to the lesion can be performed bycomparing actions of the drug on each of the above cells. In particular,the system can be utilized for searching of a drug delivery systemselective to a lesion, searching of a drug that is selectively toxic forcells in a lesion, searching of a drug that selectively terminates cellcycle of cells in a lesion and the like. Specific examples will be givenbelow. However, the present invention is not limited to these examples.

Example 5 Preparation of Liposomes

Egg PC (Funakoshi, No. 1201-41-0214), egg PS (Funakoshi,No.1201-42-0226), dicetyl phosphate (DCP, Funakoshi, No.1354-14-8165)and Hydrophobic iodine compound (3) synthesized by the method describedin J. Med. Chem., 25 (12), 1500 (1982), in the ratios described blow,were dissolved in methylene chloride contained in an eggplant-shapedflask to form a uniform solution, and then the solvent was evaporatedunder reduced pressure to form a thin membrane on the bottom of theflask. The thin membrane was dried in vacuo, then added with 1.5 ml of0.9% physiological saline (Hikari Pharmaceutical, No. 512) andultrasonicated (probe type oscillator, Branson, No.3542, 0.1 mW) for 5minute with ice cooling to obtain a uniform liposome dispersion. Size ofthe particles contained in the resulting dispersion was measured byusing WBC analyzer (Nihon Kohden, A-1042). The particle size was 40 to65 nm.

Liposome PC PS DCP Compound (3) Preparation 1 50 nmol 50 nmol 10 nmol 40 nmol Preparation 2 50 nmol 50 nmol 10 nmol  75 nmol Preparation 3 50nmol 50 nmol 10 nmol 150 nmol

Example 6 Selective Uptake of Liposome Preparations by Vascular SmoothMuscle Cells (1)

The three types of liposomes prepared in Example 5 were added to thesmooth muscle cell culture system of FIG. 1 or 2 in Example 1 accordingto the following conditions (1), (2) and (3), and then the culture wascontinued.

-   (1) A liposome preparation was added after 3 days to the culture    system of FIG. 2 not added with the foam macrophages, and culture    was continued for 1 day.-   (2) A liposome preparation was added after 5 days to the culture    system of FIG. 1 added with the foam macrophages, and culture was    continued for 1 day.-   (3) A liposome preparation was added after 7 days to the culture    system of FIG. 1 added with the foam macrophages, and culture was    continued for 1 day.

After addition of liposomes and post-culture according to each of theconditions (1) to (3) were completed, the supernatant was removed, andthe residue was washed three times with Hank's buffer (NissuiPharmaceutical, Code 05906, pH 7.2), then added with 1.5% SDS solution(Wako Pure Chemical Industries, 199-07141) and incubated at 37° C. for30 minutes to lyse the cells. Then, amount of Compound (3) taken up intothe cells was measured by HPLC. The results are shown in FIG. 5. Cleardifferences in the amounts of the iodine compound taken up into thevascular smooth muscle cells from the liposomes of the present inventionwere observed before and after the proliferation was initiated underinfluence of the foam macrophages.

Example 7 Comparative Example

An oil particle suspension of Compound (3) was prepared according to themethod described in Pharm. Res., 16, (3) 420 (1999). The suspension wasadded to the cell culture system under the same conditions as those usedin Example 6 (conditions (1), (2) and (3)) with the same amount ofCompound (3), and amounts of Compound (3) taken up into the vascularsmooth muscle cells were measured by HPLC. The results are shown in FIG.6. From comparison of the results shown in FIGS. 5 and 6, it is clearlyunderstood that, by using the liposomes of the present invention, theiodine-containing contrast medium can be more efficiently and moreselectively accumulated than the known oil particle suspension in thevascular smooth muscle cells abnormally proliferating under influence offoam macrophages.

Example 8 Selective Uptake of Liposome Preparations by Vascular SmoothMuscle Cells (2)

The three types of liposomes prepared in Example 5 were added to thesmooth muscle cell culture systems of rat (FIG. 3) and rabbit (FIG. 4)prepared in Example 4 according to the aforementioned conditions (1),(2) and (3), and the culture was continued. Amounts of Compound (3)taken up into the cells were measured by HPLC. The results are shownFIGS. 7 and 8. Each system gave results similar to those shown in FIG. 5for mouse cells.

Example 9

Arteriosclerotic lesions were formed in a rat aorta according to themethod described in Invest. Radiol. 18, 275 (1983). Liposome preparation3 prepared in Example 2 was carefully administered to the rat in whicharteriosclerotic lesions were formed in an amount of 200 mg/kg as an MTDamount converted to be an amount of Compound (3). Thirty minutes afterthe administration, clear X-ray radiographic photographs of thearteriosclerotic lesions was obtained. The results are shown in FIGS. 9and 10.

INDUSTRIAL APPLICABILITY

The liposomes of the present invention can achieve accumulation ofiodine compounds in vascular smooth muscle cells abnormallyproliferating under influence of foam macrophages, and are useful as anX-ray contrast medium for selective radiography of a lesion of avascular disease caused by abnormal proliferation of vascular smoothmuscle cells. Further, the culture systems provided by the method forpreparing a culture system of the present invention can be utilized, ascell culture systems in which a state of lesion of arteriosclerosis,restenosis or the like is reproduced, for in vitro screening of drugsand the like.

1. A liposome containing a hydrophobic iodine compound as a membranecomponent, wherein the hydrophobic iodine compound is a 1, 3,5,-triiodobenzene derivative having at least one substituent whichcontains 18 or more carbon atoms and which is a residue of a cholesterolderivative.
 2. The liposome according to claim 1, which contains a lipidselected from the group consisting of phosphatidylcholines andphosphatidylserines as a membrane component.
 3. The liposome accordingto claim 1, which contains a phosphoric acid dialkyl ester as being adiester of an alkyl containing 6 or more carbon atoms as a membranecomponent.
 4. An X-ray contrast medium, which comprises a liposomeaccording to claim
 1. 5. The X-ray contrast medium according to claim 4,which is used for radiography of a vascular disease.
 6. The X-raycontrast medium according to claim 4, which is used for radiography ofvascular smooth muscle cells abnormally proliferated under an influenceof foam macrophages.